Receptor coupled regulation of diverse cellular functions, including insulin stimulated trafficking of glucose transporters, is mediated by phosphoinositide 3-kinases that generate 3-phosphoinositide lipid second messengers. Aberrant phosphoinositide signaling has been linked to several disease states including non-insulin dependent type II diabetes. Consequently, the protein domains that bind 3-phosphoinositides are potential targets for pharmacological interdiction. The long term objectives of this proposal are to understand: i) the structural determinants of phosphoinositide recognition by lipid binding domains of cytoplasmic proteins; ii) the structural basis of protein-protein interactions at intracellular membrane surfaces; and iii) the structural mechanisms by which protein-lipid interactions cooperate with protein-protein interactions to regulate membrane localization and activation of multiprotein signaling and trafficking complexes. These objectives will be addressed through combined crystallographic, biochemical and mutational analyses of two unique model systems (Grp 1/Grsp1 and EEA1/Rab5) in which highly specific protein-phosphoinositide and protein/protein interactions co-mediate membrane localization and activation. Specifically, we will: (Aim 1) establish the structural determinants of 3-phosphoinositide recognition by the pleckstrin homology domain of the Arf nucleotide exchanger Grp1; (Aim 2) determine the structural basis for Grp1 localization and activation; (Aim 3) determine the structural basis for the interaction of the RabS GTPase with the RabS effector EEA1; and (Aim 4) determine the mechanism whereby EEA1 integrates localization signals from simultaneous interactions with RabS and phosphotidyl inositol 3-phosphate. Combined with parallel biochemical and high resolution in vivo imaging experiments of Projects 1 and 3, the conclusion derived from these studies will provide critical information regarding the structural bases and cooperative mechanisms underlying recruitment, localization and activation of multiprotein signaling/trafficking complexes that are targets of 3'-phosphoinositides.